This project addresses cellular and molecular mechanisms by which estrogens (E) and progesterone (P) regulate synaptogenesis in the hippocampus. Synapse turnover occurs naturally during the 4-5d estrous cycle of the female rat by a mechanism that requires the activity of NMDA receptors. Synapse formation has been demonstrated on CAl neurons using Golgi technique, dye filling of cells, and electron microscopy (EM). We have established a new method for demonstrating E-induced synapse formation using radioimmunocytochemistry for synaptic and dendritic markers, which this project will utilize, along with gene microarrays, to discover E and P regulation of key molecules involved in synapse formation and maturation. As far as the site and mechanism for E regulation, inhibitory interneurons may play a pivotal role, as they express the ERa receptor subtype in cell nuclei. We will test the hypothesis that inhibitory interneurons govern the excitability of the pyramidal neurons upon which new synapses are formed, and that E transiently alters both GABA and BDNF activity and allows synapse formation to occur. However, we postulate that E regulates local events in dendrites and synapses of pyramidal neurons via non-nuclear ER sites coupled to second messengers. Using EM, we have found ERa in dendritic spines, presynaptic terminals and glial cell processes. This localization is consistent with a mechanism, demonstrated by others, involving E activation of second messengers. These actions may regulate, for example, the phosphorylation of proteins involved in dendritic protein synthesis and the activity of ion channels and receptors. We shall investigate E regulation of key second messenger intermediates such as Akt and CREB at the light and EM levels, using estrogen antagonists as tools to discriminate these effects from nuclear E actions. We shall also use light and EM to study actions of P in E-primed females, since P causes down-regulation of newly formed synapses within 12-24h by a mechanism that involves intracellular progestin receptors (PR). PR are not evident in cell nuclei of the rat at the light microscopic level, but are evident in dendrites and glial cells by EM. We have also characterized ER and PR in the mouse hippocampus and have obtained preliminary evidence for E-induction of a specific marker of dendritic spines that suggests that E may promote the maturation of synaptic connections in the mouse. We plan to use the power of mouse genetics by employing mice lacking ERa and ERb to provide more definitive information regarding the role of the two known intracellular ER types in these E effects.